Organic polymers have been used as supports during polynucleotide synthesis. Early work is reviewed by Amarnath and Broom, Chemical Review, 77:183-217 (1977).
Inorganic polymers are also known in the prior art. Koster, Tetrahedron Letters, Vol. 13, 1527-1530 (1972) describes the attachment of nucleoside phosphates to silica gel using a trityl linking group. The method is apparently applicable only to pyridine nucleosides. The cleavage of the nucleoside from the silica support can only be accomplished with acid to which the purine nucleosides are sensitive.
Caruthers, et al. in Genetic Engineering, Plenum Press, New York (1982), Vol. 4, p. 1-17; Letsinger, in Genetic Engineering, Plenum Press, New York (1985), Vol. 5, p. 191; and Froehler, et al., Nucleic Acids Research, 14:5399-5407 (1986) report syntheses of oligonucleotides on inorganic solid supports bearing a succinyl linker arm. See, for example, Matteucci, et al., Journal of American Chemical Society, 103:3185-3186 (1981). See FIG. 1A. The succinyl group links the growing oligonucleotide from its terminal 3' hydroxyl group by an ester bond to a primary amine on the support, which may be controlled pore glass (CPG) or silica, by an amide bond. An oligonucleotide is freed from the support after the ester carbonyl group is hydrolyzed by concentrated ammonium hydroxide. For complete cleavage, this reaction needs approximately 3.5 hours at room temperature.
A third generation of DNA synthesizers has been developed that would not only synthesize the oligonucleotide but would also cleave the completed oligonucleotides from the support. However, waiting 3.5 hours before the DNA synthesizer can be utilized is unduly burdensome.
Further, there is growing interest in the synthesis of modified oligonucleotides possessing base sensitive functional groups as "antisense" reagents for inhibiting viral replication. Examples include methyl phosphonate derivatives, shown by Agris et al., Biochemistry (1986) 25, 6268-6275, to inhibit synthesis of vesicular stomatitis viral proteins in virus infected L929 cells and selected oligonucleotide methyl phosphotriester derivatives, reported by Buck et al., Science (1990) 24s, 208-212, to inhibit HIV-1, the causative agent of AIDS. Both the methyl phosphonates and the methyl phosphotriesters are sensitive to the ammoniacal conditions used in conventional work-up of products synthesized on insoluble supports in DNA synthesizers. Indeed, the methyl phosphotriesters are so sensitive that it has not been feasible heretofore to obtain these compounds directly from a solid support. Instead, Buck et al. employed a long, cumbersome strategy that involved: (1) automated synthesis of oligonucleotide, .beta.-cyanoethyl phosphotriester derivatives on solid CPG supports using DMT-N-protected (benzoyl and isobutyryl) nucleoside .beta.-cyanoethyl phosphoramidites, (2) cleavage from the support (succinyl anchor) by concentrated ammonium hydroxide to yield the unprotected oligonucleotide phosphodiesters, (3) reprotection of the purine and pyrimidine amino groups with 9-fluorenylmethoxycarbonyl, (4) methylation of the phosphodiester groups using methanol and toluenesulfony chloride (a relatively inefficient process), and (5) cleavage of the 9-fluorenylmethoxycarbonyl groups using triethylamine. Steps 3.congruent.5 must be carried out manually in solution after the oligomer has been removed from the support.
Another family of potentially interesting oligonucleotide analogues are derivatives with unsubstituted internucleoside phosphoramidate links (O=PNH.sub.2) Procedures for synthesis of very short oligomers of this type in solution have been reported (Tomasz, et al., Tetrahedron Letters, 22:3905-3908, (1981); Letsinger, et al, Nucleic Acid Research, 4:3487-3499 (1986); however, as reported by Tomasz et al., these compounds are sensitive to the strong ammonical conditions employed in cleaving succinyl anchors used in solid support synthesis.
There is a need for a linker arm that allows an oligonucleotide or substituted oligonucleotide to be cleaved quickly from the support under milder conditions than employed with the succinyl derivatives.
Matteucci, et al., ibid, introduced the succinyl linker arm for DNA synthesis in 1980. The linker arm is attached to the support by an alkaline resistent amide bond and to the nucleoside through an alkaline labile ester bond. In an effort to improve the cleaving ability of the succinyl group, one skilled in the art would likely increase the lability of the succinyl linker group by increasing the electron withdrawing potential of the spacer chain between the two carbonyl groups. The increased lability would result in making the carbonyl carbon more electrophilic and therefore more susceptible to nucleophilic attack by ammonium hydroxide. Schott and Letsinger in an unpublished work at Northwestern University introduced an electron withdrawing group within the hydrocarbon spacer of the linker. A diglycolyl linker arm was synthesized as shown in FIG. 1B. The electron withdrawing nature of the central oxygen atom in the spacer chain would be expected to make the carbonyl carbon more electrophilic, and thus would create a more alkaline labile ester group. It was found that the nucleosides attached to the diglycol linker arm were liberated slightly faster (35% faster) than when attached to the succinyl linker arm, but the increase was not sufficient to enable base labile oligonucleotide derivatives to be removed selectively. Simple inductive activation by the electronegative oxygen in an ether function was not sufficient.